First of all release of a oligosaccharide from the protein is necessary in the glycomic analysis of the glycoconjugate and two methods are known; one of which is to enzymatically cleave an oligosaccharide using N-glycanase, for example, peptidyl N-glycosidase F(PNGase F), and the another one is to chemically hydrazinolyze the same.
Usually the enzymatic method is preferably employed rather than the chemical method since it is possible to cleave an oligosaccharide in the enzymatic method without depending a size or structure of the glycomic moiety in the substrate. However, there are various glycoproteins and a glycomic moiety is often surrounded by the secondary and/or the tertiary structure of a protein, and according to the result an enzymatic digestion by N-glycanase is frequently hindered.
In order to improve the efficacy of digestion by N-glycanase, various methods for pre-treating a sample are proposed using a reducing agent, a surfactant, a proteinase or the combination thereof.
A reductive alkylation can be an important method for pre-treatment to simplify the complicated tertiary structure of a protein and to promote the enzymatic digestion, and in such case solubilization of a protein is carried out by adding urea and a surfactant, for example, sodium dodecylsulfate (SDS) to the solution. But it is known that MS signal may be lowered if urea and/or SDS are not completely removed from the solution.
Other important method for the pre-treatment is a fragmentation of the protein moiety using a proteinase, for example, trypsin. This method is usually applied in a combination with the reductive alkylation described above, but trypsin digestion may be hindered if urea and/or SDS are not completely removed.
Recently an acidlysing surfactant (ALS) is newly developed as an alternative of urea or SDS (See, WO03/102225). It shows an enhancing effect on digestion by trypsin or PNGase F when added as a protein solubilizer, and mass analysis is never hindered since it can be decomposed under an acidic condition after digestive reaction is over. A sulfonate salt described below is already commercialized as a trademark of “RapiGest SF”.

On the other hand, a sulfonate salt preferably used in the present invention is a kind of surfactants and use as a detergent is already known (See, WO03/082811). However, an enhancing effects on the reductive alkylation and the digestion by a proteinase described later and activity to release an oligosaccharide from glycoprotein are not known.